Investigating the Mechanisms Behind Symmetry Breaking and Cell Fate Specification in the Mouse Embryo

在所有动物物种中，最初对称的胚胎经历对称性破缺，其中胚胎的部分获得不同的身份并形成第一个细胞命运。在小鼠胚胎中，对称性在植入前发育期间的胚泡阶段（第4天）被打破，其中形成了三种不同的细胞类型：滋养外胚层（TE），原始内胚层（PE）和外胚层（EPI）-后两者形成内细胞团（ICM）。与其他模型系统不同，例如苍蝇和蠕虫模型系统，对称性破缺事件不是由卵中的分子梯度决定的，而是一个更复杂的事件。目前的模型提出：胚胎的细胞在8细胞阶段获得顶基极性，随后的细胞分裂产生外极性群体和内非极性群体。这使细胞命运偏向于分配第一不对称谱系，因为极性细胞偏向于TE，而非极性细胞偏向于ICM。我的项目侧重于形成对这个故事的更深层次的理解。首先，我正在研究早期获得顶部-基底极性的细胞所扮演的可能角色：虽然大多数细胞在8细胞阶段的“压实”事件后极化，但有些细胞在此之前极化。这种异质性对细胞命运决定具有重要影响，因为早期极化的细胞优先贡献TE。我正在研究是否与4细胞阶段的异质性有关的因素，如CARM 1（已知4细胞胚胎细胞之间的精氨酸甲基转移酶），使细胞偏向早期极化，从而以这种方式对对称性破缺产生影响。这可以使用诸如过表达和RNAi敲低的技术来实现。如果这项研究表明早期异质性和早期极化之间的联系，我将试图确定导致早期极化的分子途径和细胞机制。有证据表明，正常极化是由PKC-Rho GT3信号转导触发的，因此我将在早期极化和4细胞阶段异质性的背景下对此进行研究，例如测试CARM 1活性是否会影响PKC和/或Rho GT3活性。这可以使用诸如基于福斯特共振能量转移（FRET）的RhoA传感器的技术来实现。我也在研究决定细胞分裂模式的因素，这些因素使极性因子在8 - 16细胞阶段分裂期间得以分布。有些细胞对称分裂形成两个极细胞，有些细胞不对称分裂形成一个极细胞和一个非极细胞。我将研究各种因素，如细胞形状和顶端域的大小，影响分裂方向，以及如何早期极化细胞偏向TE通过对称细胞分裂。我可以通过荧光标记纺锤体和顶端结构域，以及测量和扰动细胞的形状和大小来研究这一点。我提出的工作将大大推进我们对小鼠胚胎对称性破缺的理解。此外，由于小鼠和人类植入前发育之间的高度相似性，这项拟议工作所产生的结果也可以揭示人类生命的起源。这可能具有与着床前发育期间经常发生的妊娠丢失相关的临床益处。M Zhu et al. Nat. Communications 8，921（2017）Q Chen et al. Nat. Communications 9，1819（2018）ME Torres-Padilla et al. Nature 445，214-218（2007）H Sasaki et al. Develop. Growth Diff 53，263-273（2010）A Takowski和J Wroblewska，Development 18：155-180（1967）M白色等人Cell 165，P75-87（2016）M Goolam等人Cell 165，P61-74（2016）CY Leung和M Zernicka-Goetz，Current Opinion in Genetics & Development 2015，34：71-76（2015）

In all animal species, the initially symmetric embryo undergoes symmetry breaking, where parts of the embryo gain different identities and form first cell fates. In the mouse embryo, symmetry is broken by the blastocyst stage (day 4) during pre-implantation development, where three distinct cell types have formed: trophectoderm (TE), primitive endoderm (PE) and epiblast (EPI) - the latter two forming the inner cell mass (ICM). Unlike other model systems, such as fly and worm model systems, the symmetry breaking event is not determined by a molecular gradient laid down in the egg, but rather is a more complicated event. The current model proposes that: cells of the embryo gain apical-basal polarity at the 8-cell stage, with subsequent cell divisions generating an outer polar population, and an inner apolar population. This biases cell fate to allocate the first asymmetric lineages, as the polar cells are biased towards TE and the apolar cells towards ICM. My project focuses on forming a deeper understanding of this story. First, I am looking at the possible role played by cells that gain apical-basal polarity early: while majority of the cells polarise after an event called 'compaction' at the 8-cell stage, some polarise before. This heterogeneity has important consequences for cell fate decisions, as cells that polarise early contribute preferentially to TE. I am investigating whether factors relating to heterogeneity at the 4-cell stage, such as CARM1 (an arginine methyltransferase known to be heterogeneous between cells of the 4-cell embryo), bias cells towards polarising early and thus have an influence on symmetry breaking in this manner. This can be accomplished using techniques such as overexpression and RNAi knockdown. If this study shows a link between early heterogeneity and early polarisation, I will attempt to identify the molecular pathway and cell machinery leading to early polarisation. There is evidence that normal polarisation is triggered by PKC-Rho GTPase signalling, so I will look at this in the context of early polarisation and 4-cell stage heterogeneity e.g. testing whether CARM1 activity would affect PKC and/or Rho GTPase activity. This can be accomplished using techniques such as a Forster resonance energy transfer (FRET) based RhoA sensor. I am also looking at the factors that determine the division pattern of cells, which enable distribution of polarity factors during the 8 - 16 cell stage division. Some cells divide symmetrically to form two polar cells, and some asymmetrically to form one polar and one apolar cell. I will investigate how various factors, such as cell shape and apical domain size, affect division orientation, and how early polarising cells are biased towards TE through symmetric cell division. I can investigate this using fluorescent labelling of spindle and apical domains, as well as measurements and perturbations of cell shape and size. My proposed work will significantly advance our understanding of symmetry breaking in the mouse embryo. Moreover, due to the high similarities between mouse and human pre-implantation development, the results yielded from this proposed work can also shed light on the beginnings of human life. This may have clinical benefits relating to pregnancy loss which occurs frequently during pre-implantation development. M Zhu et al. Nat. Communications 8, 921 (2017) Q Chen et al. Nat. Communications 9, 1819 (2018)ME Torres-Padilla et al. Nature 445, 214-218 (2007)H Sasaki et al. Develop. Growth Diff 53, 263-273 (2010)A Takowski and J Wroblewska, Development 18: 155-180 (1967)M White et al. Cell 165, P75-87 (2016)M Goolam et al. Cell 165, P61-74 (2016)CY Leung and M Zernicka-Goetz, Current Opinion in Genetics & Development 2015, 34:71-76 (2015)